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Method for knocking out Tim-3 gene to prolong survival of T cell in vivo

A TIM-3, cell technology, applied in the field of genetic engineering, can solve the problems of activation system immune risk and low efficiency, and achieve the effect of enhancing the killing function of T cells, high editing efficiency, and high efficiency transfer.

Active Publication Date: 2021-07-02
矫士平 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to provide a method for gene editing to prolong the retention time of T cells in view of the problems of low efficiency and the risk of activating systemic immunity in the prior art using antibody-directed immune checkpoint gene knockout methods

Method used

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  • Method for knocking out Tim-3 gene to prolong survival of T cell in vivo
  • Method for knocking out Tim-3 gene to prolong survival of T cell in vivo
  • Method for knocking out Tim-3 gene to prolong survival of T cell in vivo

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Experimental program
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Effect test

Embodiment 1

[0077] An overview of the T cell gene editing method, using CRISPR-Cas9 to specifically knock out the TIM-3 gene in human T cells, specifically includes the following steps:

[0078] 1. Isolation of PBMCs.

[0079] 2. Activate T cells.

[0080] 3. Preparation of RNPs.

[0081] 4. Electric transfer.

[0082] 5. T cell knockout detection.

[0083] The method of the present invention uses CRISPR-Cas9 and sgRNA to specifically knock out the HAVCR2 gene in human T cells to prolong the survival time of T cells.

[0084] Preferred technical points:

[0085] 1) gRNA sequences and combinations, by testing multiple gRNAs and their combinations, determine sequences and combinations with high cleavage activity.

[0086] 2) The process of electroporation of T cells, including determining the method of activating T cells (via OKT3+IL2) and the time point of electroporation (72h after activation). OKT3 (Orthoclone OKT3) is a baiCD3 monoclonal antibody. OKT3 reacts with CD3 on the T cel...

Embodiment 2

[0095] Preparation of tumor-specific and TIM3-T cell products (best embodiment)

[0096] 1. Isolation of PBMCs

[0097] A healthy volunteer named A, who has no symptoms of cold and fever, is recruited. 100 mL of blood was drawn from the cubital vein into BD anticoagulant blood vessels. Blood was mixed with an equal volume of PBS buffer (containing 2% fetal bovine serum). Take the PBMC separation tube Sepmate-50, add 15mL of Ficoll buffer, and then add the blood PBS mixture. Centrifuge at 1200×g for 10 minutes, then quickly pour the supernatant into a new 50mL tube, centrifuge at 200×g for 8 minutes, discard the supernatant, add 10mL PBS buffer to resuspend the pellet, discard the supernatant, add 20mL PBS buffer to resuspend After centrifuging and discarding the supernatant, resuspend all pellets in 10 mL of supernatant PBS buffer. To count the resuspended cells, take 10 μL of the suspension and add 10 μL of 0.1% trypan blue to mix well, count the cell number and surviva...

Embodiment 3

[0117] Effect of TIM3 Knockout on T Cell Retention Time

[0118] 1. Tim3-cell proliferation assay

[0119] TIM3 knockout T cells were cultured with complete medium for 1, 2, 3, 4, 5, and 6 days, and the effect of TIM3 knockout on T cell proliferation was detected by CCK8 method.

[0120] The principle of CCK8: In the CCK-8 kit, the most important ones contain WST-8, 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-( 2,4-Disulfonic acid phenyl)-2H-tetrazole monosodium salt. In the presence of electron coupling reagents (that is, cells are alive, respiration, and energy metabolism), it can be oxidized and reduced by NAD+ to form a water-soluble yellow formazan product (Formazan). The more living cells, the more Formazan produced and the darker the color.

[0121] CCK8 method was used to detect the effect of TIM3 knockout on T cell proliferation, and the test method was as follows:

[0122] 1. Prepare 100 μL of cell suspension in a 96-well plate. The culture plate was pre-i...

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Abstract

The invention relates to a method for knocking out a Tim-3 gene to prolong in-vivo survival of a T cell, in particular to a method for knocking out a TIM-3 gene of the T cell. The method comprises the following steps: preparing CRISPR-Cas9 protein and gRNA into an RNP complex, and then conveying the RNP complex into a T cell in an electroporation manner. According to the method disclosed by the invention, the survival time of a T cell is prolonged by specifically knocking out a HAVCR2 gene in the human T cell by utilizing CRISPR-Cas9 and sgRNA, so that the effect of efficiently knocking out the gene can be ensured to be realized.

Description

technical field [0001] The present invention relates to the field of genetic engineering, especially a method for gene editing to prolong the survival time of T cells. Specifically, it can be a method for prolonging the survival time of T cells by using CRISPR-Cas9 and sgRNA to specifically knock out the HAVCR2 gene in human T cells. Background technique [0002] T cell adoptive immunotherapy (car-T therapy), the full name of chimeric antigen receptor T cell therapy technology, is an emerging treatment method, which has been widely used in the field of tumor immunotherapy. It is mainly through in vitro transduction of an artificial gene that recognizes cancer cells into human T cells, so that human T cells have the ability to specifically kill tumor cells, and then introduced into the patient's body to play a specific therapeutic role. car-T cell therapy uses human immune cells to fight against tumors, which is very highly targeted. Compared with traditional chemotherapy, it...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/12C12N5/10C12N5/0783
CPCC12N15/85C07K14/47C12N5/0636C12N2310/20C12N2510/00Y02A50/30
Inventor 矫士平杨振煌
Owner 矫士平
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